Project Summary/Abstract Rational protein design is an exciting avenue for developing new vaccines, diagnostics, and therapeutics for human diseases. The ability to intentionally design thermostable proteins will allow expansion of this technique to applications that are subject to harsh storage or transport conditions. Nonfunctional, de novo designed proteins tend to be unusually stable, but there has been minimal investigation as to the structural and dynamic basis for thermostability in these proteins. It is additionally unclear whether protein designers are exploiting the same thermostabilization strategies in their designs as nature does in the proteins of organisms that evolved to live at high temperatures. We aim to determine the structural and dynamic bases for thermostability in designed proteins and compare them with thermostabilization strategies observed in thermophilic proteins. Further, we aim to determine whether the thermostabilization strategies employed by protein designers are compatible with function. We hypothesize that protein designers have employed thermostabilization strategies that are not common in nature, and these strategies may hinder designed proteins' abilities to function. The Engrailed homeodomain is a three-helix bundle transcription factor from D. melanogaster, and its structure, native state dynamics, and folding pathway have been well characterized. It served as the template in two protein design studies, both of which produced thermostable proteins. First, we will identify the thermostabilization mechanisms in these designed proteins and compare the mechanisms with those we observe in a homologous, thermophilic DNA-binding protein. Second, we will determine whether the thermostabilization strategies observed in the designed proteins are compatible with DNA-binding function. Finally, we will identify families of more complex proteins containing a designed thermostable, natural thermostable, and a thermolabile member and compare their thermostabilization strategies with those observed in the model, three-helix bundle family. These aims will be accomplished using a complementary set of computational and biophysical techniques. Molecular dynamics simulations of these proteins performed at various temperatures will generate predictions that will be tested experimentally using DNA-binding and equilibrium unfolding assays. The results of this study will provide knowledge of how to deploy thermostabilization strategies that are compatible with function, which will be critical to protein designers' success in developing protein-based vaccines, diagnostics, or therapies subjected to harsh storage conditions.